NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D...

NMR Studies on Structure and Dynamics of theMonomeric Derivative of BS-RNase: New Insights for 3DDomain SwappingRoberta Spadaccini1, Carmine Ercole2, Maria A. Gentile2, Domenico Sanfelice2, Rolf Boelens3, Rainer

Wechselberger3, Gyula Batta4, Andrea Bernini5, Neri Niccolai5, Delia Picone2*

1 Dipartimento di Scienze Biologiche ed Ambientali, Universita del Sannio, Benevento, Italy, 2 Dipartimento di Chimica, Universita degli Studi di Napoli ‘‘Federico II’’,

Napoli, Italy, 3 Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands, 4 Institute of Chemistry,

University of Debrecen, Debrecen, Hungary, 5 Dipartimento di Biotecnologie, Universita degli Studi di Siena, Siena, Italy

Abstract

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state,these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able todislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminalribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively asa mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures adimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiologicalconditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we haveanalysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studiesand Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity withRNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed alsoby H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural anddynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the knowndifferences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must beinfluenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

Citation: Spadaccini R, Ercole C, Gentile MA, Sanfelice D, Boelens R, et al. (2012) NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping. PLoS ONE 7(1): e29076. doi:10.1371/journal.pone.0029076

Editor: Piero Andrea Temussi, Universita di Napoli Federico II, Italy

Received August 12, 2011; Accepted November 20, 2011; Published January 12, 2012

Copyright: � 2012 Spadaccini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants OTKA (Hungarian Scientific Research Fund) CK-77515 and TAMOP (Operative Program for the Renewal of theSociety) 4.2.1/B-09/1/KONV- 2010-0007 (GB) (Convergence) and 20088L3BP3_001 (DP). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Protein subunits may interchange part of their polypeptide

chain, giving rise to inter-twinned dimers, or even higher order

aggregated forms. This phenomenon, also known as 3D domain

swapping, is often accompanied by additional biological activities

with respect to the parent monomeric protein, leading to new

functions or even to toxic effects associated to deposition diseases

[1]. To date, neither the molecular mechanism of 3D domain

swapping or its predictability have been elucidated (for a recent

review, see [2]).

For historical reasons [3], but mainly for the variety of

oligomers formed, bovine pancreatic ribonuclease (the well-known

RNase A) is considered a prototype of 3D domain swapped

proteins: dimers, trimers, tetramers or even higher order oligomers

of this protein have been extensively characterized, from structural

and functional point of view [4]. This protein provided also the

first experimental evidence that a single polypeptide chain may

dislocate both N- and C-termini [5,6], even in the same oligomeric

structure [7]. The capability to dislocate N-termini is a feature of

other mammalian ribonucleases [8,9], which often in the

aggregated state acquire a selective cytotoxicity towards tumour

cells [10]. However, all the multimeric forms of RNase A and

RNase-like proteins for biophysical studies have been prepared in

vitro, under non-native conditions produced by modifying either

the environmental physico-chemical conditions [11], or the

protein sequence [12,13]. Recently RNase A has been shown to

form a minor population of dimers when it is produced in bovine

cells [14]. However, only BS-RNase, which shares 80% sequence

identity with RNase A and a common catalytic mechanism [15],

offers the possibility to study the swapping process in vitro under

mild physiological conditions. The swapping endows BS-RNase

with new biological properties, including selective cytotoxicity

towards tumour cells [10]. In contrast to RNase A and all other

pancreatic-like ribonucleases, which are monomeric in their native

state, BS-RNase is a dimer constituted by two identical subunits

linked through two disulfide bridges, as well as by non-covalent

interactions. The native enzyme is isolated from seminal vesicles or

bull semen [16] as a mixture of swapped and un-swapped covalent

dimers, indicated as MxM and M = M respectively. The molar

PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29076

ratio of the two isoforms MxM and M = M is 2:1 at thermal

equilibrium. The X-ray structures of the two isomers [17,18]

revealed only small differences in their tertiary structures, located

essentially around the 16–22 hinge region, which connects the

dislocating N-terminal helix to the protein body. In contrast, there

are no differences in the quaternary structures of swapped and

unswapped forms, because the inter-subunit interface (the so-

called ‘‘open-interface’’ in the 3D domain swapping terminology

[2]), pre-exists already in the un-swapped counterpart. This

feature reduces significantly the entropic penalty associated with

the oligomerization processes. Hence in the case of BS-RNase the

dislocation of the N-termini requires only a partial unfolding step

associated with a small enthalpic barrier. As a consequence, for

this protein the swapping is a physiological, equilibrium process

[19] and the molar ratio between swapped and un-swapped forms

depends on tiny structural differences that influence the energetic

balance. Upon selective reduction of the interchain disulphide

bridges, under mild conditions [20], the swapped form of BS-

RNase retains a dimeric structure stabilized by non-covalent

interactions, whereas the unswapped one is converted into a

monomeric derivative, indicated henceforth as mBS.

A comparison of the NMR structure of mBS and RNase A,

which share more than 80% of the aminoacid sequence, showed a

close similarity between the two proteins except for the 16–22

hinge region, which has higher flexibility in mBS [21]. According

to this result and other suggestions, [22,23,24,25,26], the hinge

region may play an important role in the control of the swapping.

Inspired by these observations, we have engineered the BS-RNase

sequence by substituting either all the different residues of the

hinge loop or only the central Pro at position 19, with the

corresponding residues of RNase A. Surprisingly, none of these

variants displayed significant differences in the hinge region

flexibility and swapping extent [19,27], and the biological activity

was only marginally affected [28]. A similar behaviour has been

observed also in other swapped proteins, leading to general

conclusion that ‘‘…based on mutagenesis studies, it is doubtful that

sequence features alone determine whether a protein will undergo

domain swapping’’ [2]. On the other hand, 3D domain swapping

has been often connected to the instability of the monomeric

subunits. Therefore, we decided to perform a more detailed

investigation of the structural and dynamical properties of

monomeric derivative of BS-RNase. The in depth analysis of the

conformational stability of mBS could help to decide if the special

features of the swapping dimer arise from an intrinsic property of

its subunits, or are rather a consequence of the pre-existence of a

dimeric structure. In this paper, we report the refinement of the

structure of mBS, together with a characterization of the protein

flexibility, chemical stability, surface accessibility and hydration by

a combination of NMR techniques and Molecular Dynamics

(MD) simulations.

Results

Structure refinementThe refined structure of mBS (pdb ID code 2lfj) was calculated

out of 2252 unambiguously assigned distance constraints. These

correspond to an average of 18 restraints per residue.

The resulting structures satisfied the experimental constraints

with small deviations from the idealized covalent geometry, 98%

of the backbone torsion angles for non-glycine residues being

within the allowed regions in the Ramachandram plot. The final

structural statistics are reported in Table 1 in comparison with

those of the formerly published structure (pdb entry 1QWQ) [21].

The increased number of distance constraints makes the newly

refined structures more defined as indicated from the global values

of RMSD of the backbone and the side chains, respectively 0.730

and 1.139 A. A bundle of the 10 structures with lower energies is

reported in Figure 1A. In more detail, the higher resolution

allowed to extend the third helix by one residue and to observe a

better ordered loop spanning residues 65–72, i.e. the loop that in

the natural enzyme is involved in the deamidation of Asn 67 [29].

Despite the overall higher number of distance constraints, the

hinge loop region (residues 16–22) remains among the most

flexible regions of the protein, as indicated from the low number of

inter-residual NOEs for this region and in agreement with

previous [21,27] and new relaxation data (see below).

Regarding the three residues of the catalytic triad, His 119 is

present in almost all the structures in one conformation, His 12 is

present in two subsets of conformations whereas Lys 41 is very

disordered. This result represents a difference with respect to the

published crystal structure of the monomer, where the lysine side-

chain is bound to a phosphate ion, whereas our solution is

deprived of any salt.

Hydrogen exchange measurementsFurther information on mBS stability were derived from

hydrogen exchange experiments on 15N labeled mBS at

pH 5.65, collecting a series of 1H-15N HSQC experiments at

300 K upon dissolving in D2O a lyophilized protein sample.

Table 1. Experimental restraints and structural statistics forten lowest-energy NMR structures of mBS-RNase (2lfj).

2lfj 1QWQ

NMR distance constraints

Intra-residue, (i2j) = 0 1056 601

Short-range, (i2j) = 1 512 291

Medium-range, 1,(i2j),5 216 123

Long-range, (i2j)$5 456 260

Total distance restraints from NOEs 2240 1275

Total distance restraints from S-S bond 12 12

Total distance restraints 2252 1287

NMR dihedral restraints

W, Y 104 314

Structural statistics

RMS deviation for bond angles: 2.0u 1.9u

RMS deviation for bond lengths 0.010 A 0.010 A

Averaged pairwise RMSD (A)

Backbone (global) 0,7360,12 1.2060,55

All heavy (global) 1,1460,14 1.7360,20

Backbone (secondary structure)1 0,4560,17 0,7660,10

All heavy (secondary structure)1 0,9160,20 1,3260,44

Structure quality by procheck analysis1

Residues in most favoured regions 82.8% 72.4%

Residues in additional allowed regions 14.1% 23,9%

Residues in generously allowed regions 1.7% 2,5%

Residues in disallowed regions 1.4% 1,2%

For comparison, also the corresponding data relative to the previous calculatedstructure (1QWQ) are reported.1Residues 4–12,25–31,43–64,70–87,95–112,115–117,122–124.doi:10.1371/journal.pone.0029076.t001

Structure and Dynamics of Monomeric BS-RNase


Exchange constants could be evaluated for 45 well resolved

residues distributed all over the sequence, while the H/D exchange

process of most of the other amide groups have been only

qualitatively determined, see Table 2. Measured exchange constants

differ by more than three orders of magnitude along the sequence and

were slower by 3 orders of magnitude compared to the intrinsic rates.

For an evaluation of kop protection factors and DG based on the

exchange rates, it is important to elucidate before the mechanism

by which exchange occurs in the conditions employed for the

kinetic measurements [30]. It has been reported that under

unimolecular exchange regime (EX1) there is no pH dependence

of Kex, whereas in the bimolecular exchange limit (EX2) Kex will

be proportional to the pH [31]. Accordingly, to provide insight

into the exchange mechanism we measured the exchange rates

also at pH 6.7. The data obtained indicated for BS-RNase an EX2

mechanism, as in the case of RNase A [32].

Measured exchange rates at pH 5.65 were used to calculate

protection factors and DGop, which are reported in Table 2 and

plotted in Figure 1B. All the regions of regular secondary structure

are well protected from the solvent. As a further evidence of the

extreme stability of mBS structure, it’s interesting to notice that

also residues of some loop regions, such as residues 67–69

belonging to the deamidation loop, exchange slowly.

Relaxation measurementsConventional 15N relaxation data (T1,T2, NOE), 15N/1H CSA/

DD cross-correlated cross-relaxation rates and heteronuclear

NOEs, revealed that residues 2, 18, 20, 21, 22 and 69, 70, 71,

72 were outliers. These data confirm the earlier observations [21],

that predicted a high flexibility of the 16–22 hinge and 65–72 loop

regions. Residue 2, on the other hand, belongs to the disordered,

solvent exposed N-terminal extremity, hence the outstanding

values of its relaxation parameters. Independently of the involve-

ment of the R2/R1 outliers, the model-free evaluation [33] of the

T1, T2 and NOE data set yields tc = 7.9 ns global correlation time

and an average of S2 = 0.9060.07 for the order parameters

(overlapping signals and exchange affected residues 70–72 were

intentionally omitted from the fit). Similarly to raw relaxation data,

S2 order parameters also exhibited dips in the hinge region around

S18 (Figure 2). The back-calculated ‘theoretical’ T1, NOE and T2

relaxation data derived from these parameters agreed with the

experimental values within 5, 6 and 9% (1 standard deviation),

respectively. This modest agreement may be a sign of more

mobility, since the typical experimental accuracy of the exponen-

tial T1 and T2 fits was within 2%. Visualisation with reduced

spectral density mapping (Figure S1) [34] is a useful complement

of the model-free method. On the J(vN)/J(0) spectral density

correlation map we see that the bulk of the residues are below the

descending part of the limiting curve of the rigid body approach,

due to limited internal motions. However, residues that are

affected by exchange are right shifted because of increased J(0)

contribution (residues 70, 71 and 72). Both approaches gave a

dominant monomeric structure, with a correlation time appropri-

ate for this size of proteins. Indeed, a simple empirical formula

[35] predicts 8.5 ns correlation time at 300 K for a 124 residue

globular protein in water, that is in reasonable agreement with our

Figure 1. NMR derived structure of mBS. A) Bundle of the best 10 structures of mBS calculated out of 2252 distance constraints (pdb ID code2lfj); B) protection factors mapped on mBS structure with the following colour code: residues with a P.16105 in red, the one with 106103,P,16105

in orange and the one with P,106103 in yellow. Residues that couldn’t be analyzed are shown in grey.doi:10.1371/journal.pone.0029076.g001



findings. The small difference might be a consequence of

anisotropic rotational diffusion. 15N transversal CSA/DD cross-

correlated cross-relaxation rates again displays the same mobile

regions of mBS (see Figure S1). Combination [36] of 15N CSA/

DD cross-correlated and conventional relaxation data however,

predicted an unlikely high exchange contribution to R2 relaxation

over the whole sequence. Overall, the relaxation approach

described here unequivocally identified two mobile regions in

mBS, the hinge 16–22 and the loop 69–72 region, in good

agreement with previous heteronuclear NOE data of the same

protein and of RNase A. However, careful inspection of the NOE

(Figure S2) and S2 data anticipates enhanced dynamics of the 113–

115 turn region as well. Moreover, extra mobility can persist in the

119–121 region of the b-sheet, as it can be seen from reduced

spectral density mapping and R2/R1 rates (Figure S2). This seems

to support the curling of the 115–123 strand in the ensemble of the

refined NMR structures.

Molecular Dynamics simulationOn the basis of the refined mBS solution structure (pdb ID 2lfj) a

100 ns MD run has been performed in order to predict i) flexibility

along the protein backbone, ii) stability of intramolecular hydrogen

bonding network and iii) modes of the interaction between mBS

and water molecules. In Figure 3 rmsf values are reported for all

backbone amides, predicting the highest flexibility for the N

terminal moiety. Flexibility is also predicted for 65–68 and 111–113

fragments whose rmsf values are well above the standard deviation,

s= 1.2 A, calculated for all the residues outside the N terminus.

Surprisingly, an average rmsf value of 2.2, just above s, is observed

for the 16–22 hinge loop, that in all the experimental measurements

showed a significant flexibility. By analysing the MD trajectory,

hydrogen bond percentage lifetimes have been estimated for all

mBS backbone amide hydrogens according to previously reported

procedures [37,38]. The obtained results, compared also with the

corresponding atom depth indexes, are shown in Table 1S.

Reduced involvement in intramolecular hydrogen bonding, as

expected, is predicted only for outer backbone amide hydrogens.

Density of water molecules in contact with mBS has been derived

from the MD trajectory and the distribution of MD hydration sites,

MDHS, is also reported in Table 1S.

Urea unfoldingTo characterize the unfolding of mBS we have studied its

denaturation in a residue specific manner collecting a series of1H-15N HSQC NMR spectra at increasing urea concentration,

ranging from 1 to 7 M, and monitoring peak volumes. Figure S3

shows the spectra measured in the presence of 0, 1, 3 and 6 M

urea respectively. The HSQC spectra undergo minor chemical

shift changes at increasing urea concentration. At low urea

concentration (1 M) most of the residues of the monomeric

proteins are unaffected from the presence of the denaturant, only

the residues of the central strands show a chemical shift

perturbation higher than 0.1 ppm. However, by increasing the

urea concentration up to 3 M, peaks originating from denatured

state appear. As the urea denaturation was found completely

reversible, the simultaneous presence of peaks belonging to the

folded and unfolded state indicates slow conformational exchange

on the NMR chemical shift timescale.

The intensities of 30 residues distributed along all the sequence,

shown in Figure 4A, could be followed unambiguously up to 6 M

urea. This data indicates that still at high urea concentration there

is some localized structure. In figure 4B are shown the changes in

the normalized NMR peak intensities for selected residues across

the range of urea concentrations studied.

Table 2. Summary of Kex. protection factor (P) andDGopderived from hydrogen exchange experiments at pH 5.7.

Residue Kex(hr21)a P DGop(kcal mol21)

PHE8 0.11460.046 8.87E+03 5.4060.04

GLU9 0.27360.015 1.82E+03 4.4660.02

ARG10 0.13060.018 9.03E+03 5.4160.06

GLN11 0.02660.015 9.75E+04 6.8260.04

HIS12 0.03860.016 2.48E+05 7.3860.06

ASP14 0.19060.027 4.83E+03 5.0460.01

SER21 0.04060.100 1.56E+05 7.0160.03

CYS26 0.10960.057 4.86E+04 6.4160.02

LEU28 0.64060.073 1.15E+03 4.1860.02

MET30 0.01160.016 1.53E+05 7.0960.02

CYS31 0.13460.019 4.54E+04 6.3760.01

CYS32 2.22060.570 6.13E+03 5.1860.02

ARG33 0.24960.028 1.85E+04 5.8460.01

LYS34 0.26260.017 7.70E+03 5.3260.05

LYS41 0.20060.084 1.75E+04 5.8060.04

VAL43 0.46960.101 3.39E+02 3.4660.06

ASN44 0.03160.010 9.62E+04 6.8160.09

GLU49 0.04460.015 6.78E+04 6.6160.01

ASP53 0.72560.120 9.90E+02 4.1060.03

ALA56 0.05960.006 2.96E+04 6.1260.02

VAL57 0.00360.007 7.90E+04 6.7060.04

CYS58 0.01060.006 3.39E+05 7.5660.01

SER59 0.42560.052 2.12E+04 5.9160.01

LYS61 0.06260.075 3.11E+04 6.1460.02

GLN69 0.02660.072 8.70E+04 6.7660.04

GLN74 0.00560.014 3.43E+05 7.5760.05

SER77 0.01160.044 3.73E+05 7.6260.03

ARG80 0.19160.026 1.08E+04 5.5260.02

ILE81 0.00760.015 5.91E+04 6.5360.06

THR82 0.00560.021 1.34E+05 7.0160.05

ASP83 0.18260.025 6.19E+03 5.1960.03

CYS84 0.02760.017 1.21E+05 6.9560.04

ARG85 0.01660.015 2.88E+05 7.4760.01

GLU86 0.08360.016 8.63E+03 5.3860.09

SER89 0.03360.025 1.40E+05 7.0460.03

LYS91 0.03860.013 6.38E+04 6.5760.10

ALA96 0.62160.112 6.21E+03 5.1960.08

TYR97 0.25260.034 2.84E+03 4.7360.02

THR100 0.05760.015 3.15E+04 6.1560.06

VAL102 0.02360.010 1.85E+04 5.8360.02

LYS104 0.01460.079 5.44E+04 6.4860.04

ILE106 0.00860.013 1.61E+05 7.1260.02

ALA109 0.01460.014 6.89E+04 6.6260.07

GLY111 0.32260.086 2.21E+04 5.9460.06

VAL118 0.01260.022 1.30E+04 5.6360.02

HIS119 0.02660.012 1.66E+05 7.1460.05

VAL124 0.36060.038 3.26E+01 2.0760.04

aReported errors refer to the errors in the least square fitting.doi:10.1371/journal.pone.0029076.t002



A two state mechanism of unfolding, in which only native and

denatured molecules are considered [39], fits well with the

experimental NMR data. From the data fitting we could calculate

the concentration of urea at midpoint of unfolding (C1/2). The

selected residues show very similar values of C1/2, indicating that

all the mBS regions behave similarly as a function of urea

concentration. The average concentration of urea at midpoint of

unfolding is 4.2 M, a value very close to the one obtained

monitoring the secondary structure changes at increasing urea

concentration by far-UV CD [40]. This result definitely confirms

also that the unfolding of mBS follows a two state mechanism,

without the presence of intermediate states.

Paramagnetic profile of TEMPOL accessibility to mBSsurface

TEMPOL, the soluble and stable free radical commonly

employed for analyzing the distribution of protein surface hot

spots [41] has been used as paramagnetic probe for investigating

the surface accessibility of mBS. Changes of 1H-15N signal

intensities of backbone amides in HSQC protein spectra,

recorded in the presence of variable amounts of TEMPOL in

solution, have been measured and reported as paramagnetic

attenuations, Ai.

As shown in Table S1 Ai have been calculated for most of mBS

amide groups, i.e. 87 out of the total 118 well resolved NH signals

which are present in diamagnetic and paramagnetic 1H–15N

HSQC spectra. The obtained Ai values range from a maximum of

2.0 to a minimum of 0.4 respectively for signals exhibiting strong

and weak paramagnetic attenuations. M13, S15, T45, C65 and

G112 residues, exhibiting Ai values higher than 1.7, are the most

attenuated amide signals. Interestingly, MD simulation results (vide

supra) predict that all of them are not tightly involved in

intramolecular hydrogen bonding, see Figure 5 and Figure S4,

confirming the already suggested bias of TEMPOL to interact

with accessible backbone hydrogen bond donors [42].

Figure 2. Relaxation data of mBS. S2 order parameters of mBS as obtained from the model-free analysis of Lipari and Szabo. (Details are given inthe text.) Error bars within the 90% confidence limit were calculated with Montecarlo method Outliers according to R2/R1 rates (residues 70–72) wereintentionally omitted from the simultaneous fit of the global correlation time, effective correlation times and order parameters. The mobility of theseresidues is better demonstrated with the reduced spectral density mapping (see supplementary material).doi:10.1371/journal.pone.0029076.g002

Figure 3. Theoretical Molecular Dynamics of mBS. Root mean square fluctuations (rmsf) values vs. mBS sequence.doi:10.1371/journal.pone.0029076.g003



Discussion

Although 3D domain swapping was proposed about 50 years

ago [3], and it is actually well represented in PDB, its molecular

mechanism is still unknown. Eisenberg et al. provided its first

definition and proposed a general scheme for this process. In the

simplest case ‘‘closed’’ monomeric proteins convert themselves in

‘‘open’’ monomers through a local unfolding step. Then, the

interaction between two or more open monomers gives rise to

swapped dimers, or even higher oligomers [43]. Very recently,

Laurents and coworkers [11] proposed a different mechanism,

which involves the transition through an ‘‘activated’’, unfolded

state, re-opening the debate and the scientific interest on the 3D-

domain swapping. With respect to the proteins considered by both

authors, however, BS-RNase presents still some singularities,

which are worth of attention. Indeed, interchange of N-terminal

extremities is a physiological, equilibrium process which does not

require severe environmental conditions. The two subunits of BS-

RNase are covalently linked through two disulfide bonds, so that

the two subunits are already correctly oriented for the swapping.

In vivo, in the reducing cytosol compartment, the un-swapped

isomer is supposed to be converted into two monomers, whereas

the swapped isoform, stabilized by the interchanged N-terminal

helices, retains a dimeric structure [20]. Therefore, BS-RNase is

supposed to occur in vivo under multiple forms, depending on the

biological compartment encountered, with different tertiary and/

or quaternary structures. Given the importance of the 3D domain

swapping in the biological properties of BS-RNase, in this paper

we have performed a deeper investigation of the structural and

dynamical features in solution of its monomeric derivative, with

the aim to understand if the BS-RNase subunit has an intrinsic

feature to undergo a local unfolding and to be converted into an

open monomer, according to the Eisenberg hypothesis [44]. As a

first step, we report here the refinement of the solution structure by

Figure 4. Urea perturbation of mBS structure. A) On mBS structure are shown in red residues whose intensities were still detectable at 6 Murea; B) changes in the normalized NMR peak intensities for selected residues across the range of urea concentration studied. The line/symbol codefor each residue is the following:& K39, ¤ S77, m L32, n K26, * H12, m G111, dashed line V118, & V54, dashed-dotted line G88, ¤ D83, # E49, %- -T36.doi:10.1371/journal.pone.0029076.g004



high resolution NMR spectroscopy up to 0.730 and 1.139 A

resolution for backbone and all atoms respectively, combined with

relaxation measurements in solution at 500 MHz and with a

characterization of the surface accessibility and hydration at

atomic resolution. The increased number of restraints used for

structure calculation allowed us a better definition not only of the

secondary structure elements, -resulting in a lengthening of helix 3

by one residue- and of the side-chains, but also of the loops,

including the 65–72 region. This loop plays a remarkable role in

the biological properties of BS-RNase because it includes the Asn-

Gly sequence, which spontaneously deamidates under physiolog-

ical conditions [29], and represents also a potential contact region

with Ribonuclease Inhibitor [45], a horse-shoe shaped protein

which readily and efficiently inactivates most pancreatic-like

ribonucleases. Structural and relaxation data indicate that the

mBS behaves like a compact, globular monomer, and the

correlation time allowed us to exclude not only the presence of

dimeric forms, which instead have been observed in the

homologue human pancreatic protein [46], but also a partial

opening of the protein. Moreover relaxation data highlight the

presence of two main flexible regions, corresponding to the 16–22

and 65–72 loops; in addition, extra-mobility is found at the C-

terminal region (112–115 loop and 119–121 strand), i.e. in the

region involved in the swapping of the C-terminal strand of RNase

A [6]. A cis-trans isomerisation of proline at position 114 is involved

in this process but, despite the presence of a cis Pro in the same

position, the swapping of the C-terminal ends has never been

observed in BS-RNase.

The preferential access of TEMPOL towards few backbone

amide groups yields relevant information on mBS surface

accessibility. Indeed, the presence of backbone amide groups

which are, at the same time, surface exposed, available for

hydrogen bonding and TEMPOL accessible, as in the case of

M13, S15, C65 and G112 residues, is diagnostic of surface hot

spots, belonging to the N-terminal arm (M13, S15), the

deamidation loop (C65) and the C-terminal hinge loop (G112).

On the other hand, the high paramagnetic attenuation of T45

amide signal is consistent with the expected enhanced accessibility

of active site regions [18]. Combined analysis of data obtained

from H/D exchange and paramagnetic perturbations indicates

that amide groups of residues exhibiting the highest Ai values are

also involved in fast H/D isotopic exchange. However, fast

isotopic exchange rates do not imply strong paramagnetic

perturbations. In this respect, Y92 NH group represents a limiting

case, as it exhibits, at the same time, very fast exchange rate and

the lowest Ai value. The presence of a MDHS close to Y92 NH

could account for local presence of structured water molecules

which prevent free TEMPOL approach. It is worth noting that

high TEMPOL accessibility of S15 and C65 residues has been

already observed in the corresponding positions of the homologous

bovine RNase A in a recent multiple solvent crystal structure

(MSCS) investigation. In the latter protein, indeed, S15 and C65

residues were in close proximity of R,S,R-bisfuran alcohol and

dimethylsulfoxide respectively [47], suggesting some binding

properties of these enzyme moieties also in solution.

Thus, high compactness of the protein structure is consistently

suggested by the analysis of the H/D exchange data and

TEMPOL surface accessibility profiles, as secondary structure

elements are all protected from the solvent. This result is very

interesting and somehow unexpected, if we consider that helix 1

(residues 4–12) corresponds to the region swapping between the

two isoforms, and confirms a close similarity with RNase A

structure. In addition, the monitoring of urea denaturation of mBS

by NMR allowed us to follow the unfolding process in a residue

specific way and to elucidate, eventually, the presence of residues

more prone to unfolding and possibly prompting dislocation and

swapping. The analysis reported in figure 4 gives us a convincing

evidence of a two-step denaturation mechanism for all the protein

regions, in agreement with a recent CD and calorimetric study

[28], showing that the protein denaturation follows a two step

mechanism, and excluding the presence of significantly populated

intermediates. On the whole, all data presented in this paper

definitely confirm a close similarity with RNase A structure,

suggesting also that this protein is extremely compact and there is

no evidence of a pre-opening of the monomeric structure in

solution. With respect to our previous data [21], this more detailed

Figure 5. Tempol surface mapping of mBS. Paramagnetic attenuations, Ai, are reported for each well resolved 1H-15N HSQC signal of mBS (filledtriangles). Histogram heights refer to the fractional freedom from intramolecular hydrogen bonding predicted by the 100 ns MD simulation in explicitwater (grey bars).doi:10.1371/journal.pone.0029076.g005



investigation highlights the presence of potential hot-spots along

the protein structure, in particular in the C-terminal region,

henceforth we cannot exclude the possibility that, in different and

eventually more severe conditions, also the C-terminal swapping

could be observed. Regarding the N-terminal hinge region, this is

still one of the most flexible regions of the protein but, according to

previous mutagenesis studies [19,48], hinge flexibility together

with swapping propensity and in vitro antitumor activity [28], are

not strictly dependent on a specific sequence.

On the other hand, we cannot exclude other structural and

functional roles for the 16–22 loop, which shows a very low

similarity with the corresponding region of RNAse A. It is worth to

recall here that the presence of a proline replacing Ala 19, in the

middle of this region, prevents the subtilisin cleavage [49], and

inactivation observed in RNase [50], so that we may hypothesize

that the 16–22 hinge sequence of BS-RNase makes the protein

more resistant to protease attack. It is also worth to recall here that

in a very recent paper by Eisenberg and coworkers [51] the region

encompassing Ala 19 in RNase A has been identified as a very

good candidate to prompt protein aggregation, thus the possibility

that the substitutions present in the hinge region of BS-RNase [52]

have been selected to prevent self-assembly phenomena deserves

also consideration. For all these reason we believe that the unusual

properties of the swapping process of BS-RNase are not strictly

dependent on its N-terminal hinge region, as initially supposed,

but on the pre-existence of a dimeric structure, independently on

the swapping. With respect to RNase A the two extra disulphides

at position 31 and 32, that convert the protein into a dimer, on one

end facilitate the N-swapping, reducing the entropic disadvantage

associated to other 3D domain-swapping cases, all consisting in a

momomer/swapped dimer conversion and also preparing the two

subunits in the correct orientation, and on the other end create a

steric encumbrance which hinders the C-swapping. Finally, the

possibility that very subtle structural and dynamical differences

induced by inter-subunits interactions increase the protein

plasticity should also been considered. Further mutagenesis

studies, together with the on-going characterization of the dimers

in solution will be helpful to confirm this hypothesis and to

understand the molecular basis of the swapping process in BS-

RNase.

Materials and Methods

NMR experiments15N and 13C,15N labelled monomeric BS-RNase was expressed

and purified as previously described [21]. NMR spectra for

structure refinement were acquired at 300 K on Bruker spec-

trometers operating at 500, 600, 700 and 750 MHz, equipped

with triple resonance gradient probes. Data were processed with

NMRPipe [53]; visualization of spectra, peak-picking and analysis

were done in NMRView [54]. Chemical-shift assignments were

obtained from standard three-dimensional triple resonance

experiments recorded on 15N,13C-labeled samples of mBS [55].

NOE-based distance restraints were extracted from a 500-MHz

2D NOESY spectrum, a 500-MHz 3D NOESY-[1H,15N]-HSQC

spectrum, and a 700-MHz 3D NOESY-[1H,13C]-HSQC spec-

trum, all with a mixing time of 100 ms. 208 dihedral angle

constraints were derived from chemical shifts values using TALOS

[56]; additional restraints were obtained from 47 3JHNHA coupling

constants extracted from a 500-MHz quantitative J-correlated

HNHA experiment [57].

The urea induced unfolding of BS-RNase was performed

adding solid urea (uread4, Sigma Aldrich) directly into the NMR

tube. The urea concentration was increased by 1 M at each

titration step until 7 M. In in order to keep protein concentration

and pH constant we prepared a different protein sample for each

point of the titration and for each of them we adjusted the pH at

5.7. To check for the possible volume increase caused by the

addition of solid denaturant, the length of the NMR sample was

measured at each step and found constant (within the experimen-

tal error). The unfolding process was monitored with a series of 2D

HSQC experiments collected at 300 K.

Structure calculationStructure calculations were performed by simulated annealing

in torsion angle space, using the CYANA 2.0 package [58], which

implements an efficient protocol for structure calculation/

automated assignment of NOEs. The standard annealing protocol

was used with 20000 steps of torsion angle dynamics; in each of

seven cycles, 100 structures were calculated, and the 20 with the

lowest target function were used in the next stage. In the final run,

200 structures were computed, and the 40 with lowest target

function were refined by 4000 steps of restrained minimization in

the more realistic AMBER99 force field [59]. To mimic the effect

of solvent screening, all net charges were reduced to 20% of their

real value, and a distance-dependent dielectric constant (e= r) was

used. The cutoff for non-bonded interactions was 12 A. The

NMR-derived upper bounds were imposed as semi-parabolic

penalty functions, with force constants of 16 kcal mol21 A22; the

function was shifted to linearity when the violation exceeded

0.5 A.

The AMBER-minimized structures were sorted by increasing

restraint violation, and the first 10 were selected as the

representative bundle. Quality checks were done in PRO-

CHECK-NMR [60], while MOLMOL [61] was used for

structure visualization and analysis.

Molecular Dynamics simulationsMD simulations were performed in explicit solvent starting from

the lowest energy NMR structure of (pdb ID 2lfj). GROMACS

package [62] and the AMBER force field [63] were used for the

100 ns MD run of solvated structure in a cubic box of equilibrated

TIP3P water molecules [64]. The initial shortest distance between

the protein and the box boundaries was set to 1.0 nm and chloride

ions were added to achieve global electric neutrality. Afterwards,

the system was energy minimized with 900 steps of conjugate

gradients. To achieve a good equilibration prior to the long MD

simulations, the system was subjected to a short (20.0 ps) MD run

where the atoms of the macromolecule were restrained to their

positions, allowing only the solvent to move. The protein-water

system was simulated in the NPT ensemble by keeping constant

the temperature (300 K) and pressure (1 atm); a weak coupling to

external heat and pressure baths was applied (relaxation times

were 0.1 ps and 0.5 ps, respectively). Bonds were constrained by

LINCS algorithm, while non-bonded interactions were accounted

by using the PME method (grid spacing 0.12 nm) [65] for

electrostatic contribution and 0.9 nm cut-off for VDW contribu-

tion. An integration time step of 2 fs was used and trajectory

snapshots were saved every 0.2 ps. As the backbone RMSD levels

off after an equilibration period of 100 ps subsequent analysis of

MD trajectories was carried out from 100 ps onward. Cut-off

values of 0.35 nm and 75u between donor and acceptor moieties

have been considered for hydrogen bonding. The fraction of the

MD trajectory in which hydrogen bond criteria are fulfilled for a

given donor is denoted by ‘‘hydrogen bond percentage life time’’

[37,38]. Solvent density map, whose maxima have been defined as

molecular dynamics hydration sites (MDHS’s) [66,67], were

calculated from the atomic coordinates of the explicit waters in



the simulation. The space surrounding the protein was divided

into two shells: the first one extended up to a distance of 0.6 nm

from the protein surface and accounted for the protein hydration

sites; the second one, representing the bulk solvent, extended from

0.6 nm to 0.8 nm from the protein surface. The water positions

were accounted in a 3D grid (step-size 0.05 nm). For each frame,

the protein was superimposed onto a reference structure in order

to eliminate the effects of translations and rotations. 3D iso-

contour plot of the resulting water density was obtained by using

PyMOL software (http://www.pymol.org).

Hydrogen exchangeHydrogen exchange experiments were performed dissolving the

15N-labelled lyophilised NMR protein sample in 99.9% D2O. A

series of successive 2D 1H-15N HSQC spectra was recorded and

300 K. Fifty hydrogen exchange rates were determined by fitting a

first order exponential equation to the peak heights versus time data:

I(t)~I(0) exp ({kobst)

where I (t) represents the cross peak height at time t, I(0) the original

cross peak height, kobs the observed rate of hydrogen exchange and t

the time in seconds. The intrinsic hydrogen exchange rates krc were

calculated using the web server program Sphere (http://www.fccc.

edu/research/labs/roder/sphere, Yu-Zhu Zhang, Protein and

peptide structure and interactions studied by hydrogen exchange

and NMR. Ph.D. Thesis, Structural Biology and Molecular

Biophysics, University of Pennsylvania, PA, USA.). The protection

factor (PF) for each well resolved NH group was then calculated

using the equation:

PF~krc=kobs

The free energy of opening, DGop, was calculated from the equation

DGop~{RTlnKop

where kop = kobs/krc. Data were fitted with the program Kaleida-

Graph 3.51 (Synergy software) [68].

RelaxationFor NMR dynamics, besides the conventional 15N relaxation

(T1, T2 and 15N-NOE) [69], the longitudinal and transverse 15N

and 1H CSA/DD cross-correlated relaxation rates gzz(15N),

gxy(15N), gxy(

1H) [46], were measured at 500.13 MHz on a

Bruker DRX spectrometer. To this end, a series of 2D 1H-15N

HSQC spectra using sensitivity enhanced gradient pulse schemes

[70] were recorded. The typical experimental parameters are

summarized as follows. The 1H carrier frequency was set to the

water resonance at 4.7 ppm using 15 ppm window, while the 15N

window was 28 ppm centred at 118.5 ppm. The relaxation delay

times were set as follows for T1: 11.2, 101.2, 201.2, 401.2, 601.2,

801.2, 1001.2, 1201.2 ms; for T2, CPMG pulse trains of 0.03,

30.4, 60.8, 91.2, 121.6, 182.4, 243.2, 302.4, 360, 417.6 ms in

duration were used; for the measurement of cross-correlated

relaxation rates, gzz and gxy, the relaxation interference was

allowed to be active for 20 or 21.6 ms in pairs of experiments

including the reference (D= 0 ms) experiment. The number of

transients collected per t1 increment was 8 for T1, 16 for T2, 32

for NOE, 128 for gzz and 80 for gxy measurements. A spin-lock

field of 3400 Hz was used for the 15N transverse cross-correlation

experiment. Two-parameter exponential fits of the measured

volume intensities of cross-peaks were applied to extract the

relaxation times T1 and T2. The cross-correlation rate constants

were determined using the initial linear build-up rate approach.

The theoretical expressions for the autorelaxation (R1, R2) and

cross-correlation rate constants (gxy, gzz) and for the steady-state

heteronuclear NOE in terms of the spectral density functions

(Ja(v) auto- and Jc(v) cross-correlation) are used as given in the

literature [71]. The simplifying assumption of isotropic overall

tumbling and the axial symmetry of constant (Ds= 2160 ppm)15N chemical shielding tensors were applied. A bond length of

rNH = 0.102 nm was used in all calculations. The model-free [33]

analysis of T1, T2 and heteronuclear NOE yielded the S2 order

parameters and local correlation times for most amides and the

global correlation time.

Paramagnetic studies of Tempol induced perturbations1H–15N HSQC diamagnetic and paramagnetic spectra, record-

ed from a Bruker DRX 600 spectrometer with 512 increments and

128 scans over 2048 data points, were compared to determine

paramagnetic perturbations on signal intensities.

Only well resolved NMR signals have been considered for

quantifying cross-peak volumes in the presence and in the absence

of the paramagnetic probes, respectively Vid and Vi

p. Such volumes

have been measured with an estimated error lower than 10% and

their autoscaled values, ui, were used according to the relation:

up,di ~

Vp,di

(1=n)P

n

Vp,di

Paramagnetic attenuations, Ai, were calculated from the auto-

scaled diamagnetic and paramagnetic peak volumes, respectively,

ud and up, according to the relation:

Ai~2{u

pi

udi

Atom depth calculationsBy using the SADIC suite [72], atom depth of all the hydrogen

of mBS backbone amide groups have been calculated. A radius of

8 A for the probing sphere was employed to measure exposed

volumes and, hence, depth indexes, Di.

Supporting Information

Table S1 Experimental and calculated parametersobtained for solvent-protein interactions.

(DOC)

Figure S1 Relaxation data of mBS. A) Residue by residue

representation of 15N spectral densities as a function of low-

frequency components. The continuous curve represent a rigid

body, with 8 ns global correlation time. The outliers to the right of

the curve clearly identify residues 70–72 as the most exchange

affected part of mBS; B) Transversal 15N-1H CSA-DD cross-

correlated cross-relaxation rates of the amides in mBS. Lower rates

can be attributed higher internal mobility.

(TIF)

Figure S2 Relaxation measurments on mBS. A) Hetero-

nuclear 15N-{1H} NOE values in mBS; B) Ratio of 15N T1/T2

relaxation times in mBS.

(TIF)



Figure S3 Urea denaturation experiments. 1H-15N

HSQC spectra of mBS at different urea concentrations.

(TIF)

Figure S4 Surface accessibility of mBS. Correlation

diagram between TEMPOL-induced paramagnetic perturbations

and fractional HB freedom predicted by MD simulation. The least

squares linear fit, represented by the straight line, yielded the

following parameters: slope 0.22, intercept 0.81, correlation

coefficient 0.18.

(TIF)

Acknowledgments

A special thank is due to Dr. Teodorico Tancredi for his help and constant

encouragement.

Author Contributions

Conceived and designed the experiments: DP RB RS NN GB. Performed

the experiments: RS CE AB RW GB DP. Analyzed the data: RS MAG DS

AB GB NN. Contributed reagents/materials/analysis tools: DP RB NN

GB. Wrote the paper: DP RS NN GB.

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NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D...

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Transcript of NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D...